The Effect of Oxygen and Carbon dioxide 
On the Development of Certain 
Cold Blooded Vertebrates 


By 


Ada Roberta Hall 


A. B. University of Oregon, 1917 
A.M. University of Oregon, 1919 


AY DIGEST-OP A THESIS 


SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY IN THE 
GRADUATE SCHOOL OF THE UNIVERSITY 

OF ILLINOIS, 1921 


Reprinted from Ecology Vol. V, No. 3 p. 290 and 
Ecology Vol. VI, No. 2 p. 104 


Digitized by the Internet Archive 
in 2022 with funding from 
University of Illinois Urbana-Champaign 


https://archive.org/details/effectofoxygenca0Ohall_0 


abt 


[Reprinted from Eco.oey, Vol. V, No. 3, July, 1924.] 


THE. EFFECT OF OXYGEN AND CARBON DIOXIDE ON THE 
DEVELOPMENT OF EGGS OF THE TOAD, 
BUFO AMERICANUS? 


Apa R. Hat 


University of Illinots 


INTRODUCTION 


The success of a species in a given habitat depends largely upon the part 
which environment plays in the development of the individual, and the loca- 
tion of the most sensitive periods in the life history. The toad, Bufo 
americanus Le Conte, lives and breeds in warm, temporary, stagnant pools, 
thick with suspended mud and decaying matter, subject to many fluctuations 
in temperature and all other environmental factors, while the whitefish,” 
must have clean cold water comparatively free from decaying materials, pre- 
senting the minimum of daily fluctuations. This paper is an attempt to deter- 
mine the effects of variations in dissolved gases, of a magnitude common in 
the toad habitat, upon development, and to locate the sensitive stages in the 
early life history. An index of sensitivity was also sought in the amount of 
oxygen released from hydrogen peroxide. 


MATERIALS AND METHODS 


It was found that the toads at Urbana, Illinois, breed in a variety of 
places from barnyard pools thick in manure and mud, to temporary pasture 
puddles. A plentiful supply also breed in a small artificial lily pond on the 
campus of the University of Illinois where the adults may be easily captured. 
They begin laying early in the morning and will continue until noon or after 
when brought into the laboratory. This affords a continuous supply of 
freshly fertilized eggs throughout the morning, and is more satisfactory than 
artificial insemination. A further advantage that these eggs possess for such 
a study as this is the fact that the time from fertilization to the functioning 
of the internal gills at 16° C. is very short, about twenty days. This makes it 
possible to watch their growth from hour to hour and to see the effect of 
changing the conditions. The eggs are large enough to see the stages well 
under a hand lens. 

The pH was determined by the use of Hynson, Westcott and Dunning 
indicator sets with additional tubes for high and low values. The oxygen 
determinations were made by the Winkler method. As the amounts of 


Contribution from the Zoological Laboratories of the University of Illinois.244 
2 Studied for comparison, results to be published later in FcoLocy. 


290 


No1833 


( 


291 ADA R. HALL Ecology, Vol. V, No. 3 


B 
it! ip - / 


Fic. 1. Apparatus for measuring the oxygen content of small amounts of water by 
the Winkler method. A, large tube 2.2 cm. diameter by 6.5 cm. length, capacity 26 cc., 
total length from I to 2 is 17 cm. R to R heavy black rubber tubing enclosing glass 
rings B C and D, diameter I cm. outside, .6 cm. inside. 


July, 1924 EFFECT OF OXYGEN ON THE TOAD 292 


water in the experimental dishes were small, 200 to 400 cc., the use of 250 
cc. bottles was impracticable. A piece of apparatus was therefore devised 
that would handle small amounts of water (Fig. 1). By raising and lowering 
the mercury cup, water was siphoned into the apparatus from the experi- 
mental dish, and the cocks 1 and 2 closed. B was filled with manganous 
chloride (sol. I, Winkler method), C with potassium hydroxide-iodide (sol. 
IT), and D with concentrated hydrochloric acid, and clamps 3, 4, and 5 
closed after each addition. The clamps were then removed one at a time in 
the same order and the liquids mixed by means of gravity, and a glass bead 
inserted with solution I. The 26 cc. of water which the apparatus holds was 
then titrated against N/4o sodium thiosulphate and the oxygen in cc. per 
liter calculated. 
. EXPERIMENTAL DATA 


A. Physiological Life History from Fertilization to Internal Gill Stage 


In this work on physiological life history several types of experiments 
were performed: (1) Keeping the pH constant (8.0) the oxygen content 
was varied, four concentrations, .4 cc., .9 cc., 1.41 cc. and 4.64 cc. per liter 
being used. (2) Keeping the oxygen about the same in all dishes, .4 cc. to 
.8 cc. per liter, the pH was varied from 6.1 to 8.0. (3) Eggs were sealed into 


abcd ¢€ fghi jy k om n ° Paodtes t BDicds Cel teh) Tal} wen n S ahs 1s 


1. I 


Fic. 2. Toad eggs put into experimental conditions: I at 1 cell stage, II 2 cell stage, 
and III 4 cell stage. 

a fertilization, b 2 cell, c 12-16 cell, d early blastula, e late blastula, f blastopore lip, 
g yolk plug large, h yolk plug medium, i yolk plug small, j early neural folds, k late 
neural folds, m elongation, n tail differentiating, o tail 1/4 body, p tail 1/3 body, r tail 
1/2 body, s tail 2/3 body, t tail equal to the body (7 days), u 8 days, v 9 days, x point 
of death. 


293 ADA R. HALL Ecology, Vol. V, No. 3 


known quantities of water with differing oxygen content and the time and 
extent of development noted. 

Time for normal development of toad eggs at 16° C., plotted as a 
straight line in graphs of figs. 2 and 3, is as follows: Fertilization o hrs.; 
first cleavage (2 cell), 3 hrs. and 20 min.; 4 cell, 4 hrs. and 30 min.; 8 cell, 
6 hrs.; 12 to 16 cell, 8 hrs.; early blastula, 10 hrs. and 30 min.; late blastula, 
23 hrs. and 30 min.; blastopore lip, 28 hrs.; yolk plug-large, 31 hrs: and 30 
min.; yolk plug medium, 41 hrs.; yolk plug small, 48 hrs.; early neural folds, 
51 hrs.; late neural folds, 62 hrs.; elongation, 72 hrs.; out of jelly—tail dif- 
ferentiating, 95 hrs.; tail 1/4 length of body, 114 hrs.; tail 1/3 length of 
body, 123 hrs.; tail 2/3 length of body, 143 hrs.; tail equal to body, 168 hrs.; 
gills three, 192 hrs. — 


TasLe I. Time in hours for reaching various stages of development in 
the toad experiments 


Read down the columns; embryos died where the number is underscored. 


Series I. Put in 25 Min. Series II. Put in at 
before Ist Cleavage 2 Cell Stage 
A-1 | A-4 | C1 C-2 A-1I A- GI C-2 
1 Sythe 3 eects eae 8.0] 8.0 | 5.8-6.6 | 6.0-6.4 8.0 8.0 |5.8-6.6 | 6.0-6.4 
Oxygen Content (c.c.).. 4| 4.64 8 4 4 4.64 8 4 
Time from fert. to: 
TStACleaVage ee es eis 2254s. 3.5 Be 5 3.5 3.5 25 3.5 
Farly ‘blastula ie sacs See BAe eats Mee | utiee de @ Ieee are: Nate 24 24 
Late blastula........ 24 56 24 24 24 24 (Sate Ae SO 
Blastopore lip....... on Woah ode 56 56 60 5On) § | Senet leeeem sere 
Wolk plug: larve (eee sock), ey ed eee ot oe ee ene 734 ll, Sage sce Seer 73 
Wolk plug medians enogi ss... s| ease FB 0 | Sisiete! choos | Saetere cea chee ee | peers rete ae Haas 
May neural foliage. | o..-< oem hee Pte 7 3 te eee hs RS eo ath pol is conse 
ote neural folds act 73. |ncen eee 95 95 Zien Reeds PR Cis abies” 
Blonyation 15ee6. eee tie Tew led nee 123 123 Oot, doen eam 
ail iditter peers 95 else Aletsic is So cal Se od py) Mian Wilbon sea aso. 
Jail 4 body: the roc} 123 “YI23 | case ae ee Prodi awehS tale 2 Se alle doers: 
Maileyasbodyacrencroe 1 be Mega eee Pept Mie UE A deka te ce |i il Read UAONET cto htatod. coco 
Tail ibody..ea.4: TOSP ph TAS Ti aega el cle iene oc eet LOB Me. eae ee atioe Lae 
Taylt24 ibody aaarnac LOZ) | LOB. ei cee APA eer 5 ees reiesetel cliche ete | ceca cts gee 
Cail s=sbody oi aise s als Fea 64 LO QUI fd nate aie ole a ate heptiene wher sllhe 2s inc eee 


In the first type of experiment eggs were put into the experimental dishes 
at different stages of development, and the effects as shown by retardation, 
acceleration or death noted. These results were tabulated in Table I, while 
the A series and the same results are shown graphically in figures 2 and 3. 
In making the graphs, normal development was taken arbitrarily as a straight 
line from fertilization at o hours to the internal gill stage at 196 hours. 
Time in hours was plotted as ordinates, and the stages of development, or 
abscissae, were obtained by dropping perpendiculars from the normal de- 
velopment line to the + axis according to the number of hours required to 


July, 1924 EFFECT OF OXYGEN ON THE TOAD 294 


reach that development. All experimental curves were then drawn with both 
time and period of development fixed by the normal curve. When the ex- 
perimental curve rises above the normal line retardation is indicated; when 
it drops toward the normal or below it, acceleration is indicated. Curves 
parallel to the normal denote normal rate of growth. 


= 


ab cd Cn tig hell a m n ° Pp 
0 F 


Fic. 3. Toad eggs put into experimental conditions: I at late blastula, II at yolk 
plug, and III at blastopore lip stages. Notation same as Fig. 2. 


Using a pH of 8.0 and four concentrations of oxygen, .4 cc., .9 cc., 1.41 
cc., and 4.64 cc. per liter, we find that a certain degree of development was 
attained regardless of the concentration, death not occurring in any of the 
experiments short of the yolk plug stage even when the oxygen was as low 
as .4 cc. per liter. Eggs put in before the first cleavage (Fig. 2-I) show 
marked retardation up to the late blastula stage then some acceleration so 
that further development approaches normal. Eggs exposed to the experi- 
mental conditions between the first cleavage and the late blastula stage (Figs. 
2 and 3) show a period of eighteen or twenty hours with little or no retarda- 
tion and then a period of quite marked retardation occurring in the blastopore 
or yolk plug stages. Later development for the higher concentrations of 
oxygen again approaches normal. Eggs put in after the late blastula stage 
(Fig. 3) show general retardation for the lower oxygen concentrations but 
fairly normal development for the higher concentrations. 

The results of this type of experiments show that where the oxygen was 
as low as .4 cc. to .9 cc. per liter death occurred in part of the experiments, 
but at higher concentrations only retardation showed the effects of exposure. 
At the late blastula and blastopore lip stages the higher concentrations even 
caused acceleration of growth. The most sensitive stages appeared to be 
from fertilization to the first cleavage, and the gastrulation stages. 


295 ADA R. HALL Ecology, Vol. V, No. 3 


_ The second type of experiment was where the oxygen was low (.4 to 8 
cc. per liter) and the pH varied, 6.1, 6.4, and 7.0 to 8.0. The stages reached 
in development are shown in the graphs of Figs. 2 and 3. Exposures to these 
conditions beginning before the four cell stage gave death with retardation 
in 24 to 120 hours, c to k. The same effect is seen in eggs put in after the 
eight cell stage. Those exposed between the four and eight cell stages show 
the greatest development (198 hrs.) which is past the sensitive stages. With 
small amounts of oxygen the effect was much more rapid and deadly with a 
pH of 6.1 to 6.4 than with a pH of 6.4 to 6.8. 

In the third type of experiment a dozen eggs were placed in an eight 
dram vial (about 26 cc. of water) supplied with a two-holed stopper having 
the inlet tube extended to the bottom of the vial and the oulet tube even 
with the stopper. Both tubes ended outside the stopper in short rubber 
tubes. Water of the desired oxygen content was run through the bottle 
until all oxygen due to air was flushed out and then a clamp on the two rubber 
tubes sealed the bottle. Development was watched by means of a dissecting 
lens until growth stopped. The oxygen content was then determined by 
adding the chemicals through the inlet tube, being careful to avoid air bubbles 
when inserting the pipettes in the rubber tubes. The cleavage of a dozen eggs 
proceeded to the late blastula stage regardless of the period of development 
when put in or the amount of oxygen present (.1-5.7 cc. per liter). Further 
development was roughly proportional to the amount of oxygen present, as 
was also the time to death. 


B. Reaction of Eggs to Hydrogen Peroxide as Development Progresses 


A universal property of protoplasm (both plants and animals) is the 
abitilty to liberate oxygen from hydrogen peroxide. What this property is 
due to is a debated question at present, but in general it is ascribed to an 
enzyme called catalase. Several theories have been advanced as to its sig- 
nificance in animal life. References are given to these in the literature 
cited (Becht ’19, Burge and Burge ’21, Zieger ’15). As a measure of the 
sensitivity of the developing egg at different stages in the early life cycle the 
reactions to oxygen and carbon dioxide (acidity) have been discussed. It 
was thought that a study of the amounts of oxygen released from hydrogen 
peroxide at various periods of development might be of significance. Ac- 
cordingly a series of determinations was made with the toad egg using the 
method outlined by Burge (16), Fig. 4A. A second series was made for the 
toad eggs the following spring using eight dozen eggs instead of one dozen 
and shaking the material by machine instead of by hand (Fig. 4B). A com- 
parison of A and B shows a striking parallelism and the two curves seem to 
confirm each other. 

From a study of these curves (Fig. 4) it may be seen that there was a de- 
crease in the power of liberating oxygen from hydrogen peroxide from the 


July, 1924 EFFECT OF OXYGEN ON THE TOAD 296 


unfertilized egg to the fertilized, an increase from fertilization to the early 
blastula, a decrease from early blastula to elongation of the embryo, and from 
this point to twenty-one days a steady increase. The stages most sensitive 
to low oxygen and high acidity were (1) fertilization to first cleavage, and 
(2) the gastrulation stages. These were the low points on this curve. Is 
there a correlation between sensitivity and a lowered power of liberating 
oxygen from hydrogen peroxide? 
| 


| 
cc icslonsed by 


200(10 
18049 
160}8 


10075 | ae 
: Samat eel 

8074 | pe sag ec | 

6073 - 


ov ab cd Sur ehh ey key im n ° p r s t u v 


Fic. 4. Amounts of oxygen released from hydrogen peroxide at different stages of 
development of toad eggs. Series A determinations were made with one dozen eggs in 
1920. Series B was made using eight dozen individuals in 1921. 


Winternitz and Rogers (’10) have shown a definite increase in what they 
call catalase for the different stages of the hen’s egg, as development pro- 
ceeds. Burge and Burge (’21) have shown an increase in this power from 
egg to adult for the Colorado potato beetle. Zieger (’15) has found a definite 
rythm for catalase in the insect life history, reporting that the power is high 
where rapid growth or metamorphosis is going on (early larval stages, 
pupal stages) and low during resting stages. Child in his book “ Senescence 
and Rejuvenescence’”’ has shown that metabolism is high during the more 
sensitive stages. Burge believes that increase in catalase brings about an 
increase in metabolism. This curve for the toad egg seems to be a contradic- 
tion of one statement or the other since it shows the power of splitting 
oxygen from hydrogen peroxide to be low at the sensitive stages. More 
work is needed on complete life histories before definite conclusions can be 
drawn about this point. 

I wish to thank Dr. V. E. Shelford, at whose suggestion this work was 
undertaken, for his helpful advice and kindly criticism during the course of 
the investigation. I am indebted to Dr. W. E. Burge and Dr. H. B. Lewis 
for suggestions in the enzyme work. 


297 ADA R. HALL 


SUMMARY AND CONCLUSIONS 


1. Conditions of high carbon dioxide or low oxygen content such as may 
occur in the normal environment and which are tolerated by the other periods 
of development, cause great retardation and even death at sensitive stages. 
The effects of low oxygen are much more detrimental in acid than in alkaline 
water. 

2. There are two definitely sensitive stages in the early physiological life 
history of the toad (a) one cell stage; fertilization to first cleavage, (b) the 
gastrulation stages; blastopore lip formation to the small yolk plug stage. 

3. There is a decrease in the amount of oxygen released from hydrogen 
peroxide at the sensitive periods which roughly corresponds in magnitude to 
the decreased resistance. 


LITERATURE CITED 


Becht, F. C. 1919. Observations on the Catalytic Power of Blood and Solid Tissue. 
Am. Jour. Physiol., 48: 171-1901. 

Burge, W. E. 1016. Relation between the Amount of Catalase in the Different Muscles 
of the Body and the Amount of Work Done by these Muscles. Amer. Jour. 
Physiol., 41: 153-161. 

Burge, W. E., and Burge, E. L. 1921. An Explanation for the Variation in the In- 
tensity of Oxidation in the Life Cycle. Jour. Exp. Zool., 32: 203-206. 

Coventry, A. F. 1011. Note on the Effect of Hydrochloric Acid, Acetic Acid, and 
Sodium Hydroxide on the Variability of the Tadpole of the Toad. Arch. Entw. 
Mech., Band 31: 338-341. 

Hall, A. R. 1918. Some Experiments on the Resistance of Sea-urchin Eggs to Sul- . 
phurous Acid. Pub. Pug. Sd. Biol. Sta., 2: 113-1109. 

Jenkinson, J. W. 1010. The Effect of Sodium Chloride on the Growth and Variability 
of the Tadpole of the Frog. Arch. Entw. Mech., Band 30: 349-356. 

Winternitz, M. C., and Rogers, W. B. t1g10. The Catalytic Activity of the Developing 
Hen’s Egg. Jour. Exp. Med., 12: 12-18. 

Zieger, Rudolph. 1915. Zur Kenntnis der Katalase der neiderer Tiere. Biochem. 
Zeitschrift, 69: 39-110. 


{Reprinted from Ecoxocy, Vol. VI, No. 2, April, 1925.] 


EFFECTS OF OXYGEN AND CARBON DIOXIDE ON THE 
DEVELOPMENT OF THE WHITEFISH? 


Apa R. Hatt 
University of Illinois 


Introduction 


A subject of vital interest is the rhythm of events in the physiological life 
history of any species, and the manner in which this rhythm may be affected 
by environmental factors. Accordingly this work has been undertaken for 
the purpose of: (1) finding out the relative sensitivity of the stages in the 
early life history of the Whitefish; (2) testing the resistance and reactions 
of normally hatched individuals as compared with the reactions of those 
hatched under experimental conditions. 


Materials and Methods 


The material for this work was the lake whitefish, Coregonus clupeiformis 
Mitchill. Some work was done on fertilization and early cleavage stages at 
the U. S. Hatchery, Put-in-Bay, Ohio. The major part of the work, how- 
ever, was done at the Vivarium, University of Illinois, on material shipped 
from the hatchery in ice. The temperature of the lake water was about 8° 
C. at the beginning of the season. It had a pH of 7.0 and an oxygen content 
of 4.08 cc. per liter. At the Vivarium the stock was kept in water from the 
University wells aerated to about 2.6-3.3 cc. per liter, and with a pH of 7.8. 
It was cooled to 10° C. by means of brine coils. The water (pH 8.0-9.0; 
carbonates 232 + parts per million) for all the experiments was boiled free 
of all dissolved gases, and part of the salts precipitated (Shelford ’18). 

The apparatus used for varying the pH and oxygen content is shown in 
figure I. Bottle 1 contained approximately N/4 sulphuric acid which 
siphoned over into the mixing bottle 4. The stopcock 2 controlled the flow 
which could be measured by counting the drops through the glass bulb 3. 
Thus a known amount of the acid was added to a known flow of the boiled 
water which entered at W. This water was also 10° C. The water leaving 4 
had a known pH, and was oxygen free. Since it contained carbonates, acids 
set free carbon dioxide which changed the pH. The oxygen was controlled 
by adding compressed air (see fig. 1 and explanation). Three such sets of 
apparatus were used giving three hydrogen ion concentrations, each with 
three oxygen concentrations. 

1 Contributions from the Zoological Laboratory of the University of Illinois, No. 256. 

104 


105 ADA R. HALL Ecology, Vol. VI, No. 2 


The pH was determined by the use of Hynson, Westcott, and Dunning 
indicator sets with additional tubes for high and low values (Clark ’20). 
Brom cresol purple (5.8-6.6), brom thymol blue (6.6—7.6) phenol red (6.6— 
8.0), and thymol blue, alkaline range (8.0-9.2) were used. The colorimetric 


nee 


alias 


Fic. 1. Apparatus for the control of experimental conditions. Method of varying 
hydrogen-ion concentration and oxygen content. 1, N/4 sulphuric acid; 2, cock for con- 
trol of acid by drops at 3; 4, mixing bottle for acid and boiled water from W; 5, 6, and 
7, jars, to which air from A is added in varying amounts; C, cocks for control of air 
flow; 8, 9, and 10, half-pint sedimentation glasses for eggs; M, mercury manometer for 
keeping air flow constant. 


standards were checked electrometrically by Dr. R. E. Greenfield. Oxygen 
determinations were made by the Winkler method with an apparatus which 
used only 26 cc. of water for a determination (Hall ’23). 


Experimental Data 
EXPERIMENTS AT THE HATCHERY 


All the experimental work was done at 10° to 11° C. at the U. S. Hatch- 
ery. The length of life of eggs and sperm was tested. Three sets were run 
(a) dry eggs and sperm, (b) dry eggs and sperm mixed thoroughly with 
water to uniform milky fluid, and (c) dry sperm and eggs standing in lake 
water. With wet eggs and dry sperm eight minutes was the latest time at 
which fertilization was possible. With wet sperm and dry eggs nine minutes 
showed a small number of fertile eggs. But with both eggs and sperm dry 
fertilization took place at seven and a half hours. It may have been possible 
after longer time as such an experiment was destroyed and could not be veri- 
fied. 


April, 1925 DEVELOPMENT OF THE WHITEFISH 106 


In early development the eggs were exposed to constant oxygen (4.08 cc. 
per liter) but with varying pH. Normal lake water had a pH of 7.0. 
Acidity (pH 6.2-6.6) was produced by adding sulphuric acid, and alkalinity 
(pH 8.4-8.6) by adding sodium hydroxide. Dishes of standing water were 
used and changed frequently. Eggs fertilized directly in the solutions were 
normal at the end of twenty-four hours (32-64 cell stage). This experiment 
was then repeated using lake water boiled until the oxygen content was 2.9 
cc. per liter. Fertilization and development occurred in all the dishes, but 
there was a marked difference between the acid and alkaline waters. With 
a pH of 6.2 and 6.6, respectively, 80 per cent and 25 per cent fertilized and 
developed; with a pH of 7.0 only 3 per cent, and at a pH of 8.4 but I per 
cent. A hydrogen ion concentration which is too great to favor later de- 
velopment appears best for fertilization (Cohn 718). 


EXPERIMENTS WITH SHIPPED Eccs 


In the work on later stages with treated running water at the University 
of Illinois, special attention was paid: (1) to keeping conditions as near 
constant as possible from day to day; (2) to watching the stages of develop- 
ment reached in each concentration and comparing these with each other and 
the control stock; (3) to working out the death rate and the percentage 
hatching for each concentration; and (4) to testing the vitality and reactions 
of the larvae hatching from the different stocks. 

In this work apparatus was set up in triplicate (fig. 1). Values of pH 
6.4, 7.0, and 8.0-9.0 respectively were used, each pH with an oxygen content 
of I cc., 3 cc., and 4.5 cc. per liter; pH was taken in all the dishes each day, 
and oxygen was determined every two or three days. 

The eggs were obtained from the hatchery in four lots. The first lot, 
spawned December 2, was in the thirty-two cell to early germinal cap stage 
when received December 3. The second lot, spawned December 5 and re- 
ceived December 24, had the tail just elongating and the fin buds starting. 
The eye vesicles had formed but no pigmentation had occurred. The third 
lot, spawned December 7 and received January 31, were only slightly far- 
ther along in development than lot two. They had just begun to show the 
pigment in the eyes. The fourth lot, recetved March 12, were fully devel- 
oped and started to hatch immediately. 

A comparison of the chemical analyses of Lake Erie water (the average 
of Huron at Port Huron and Erie at Buffalo, Clarke ’20, p. 70) and the 
boiled University water is important in connection with the experimental re- 
sults obtained with the whitefish, and is given in Table I. 

The four stocks differed in: (a) the length of time in these two kinds of 
water, (b) temperature at hatchery and in experiments, and (c) the stage at 
which shipment was made. 


107 ADA R. HALL Ecology, Vol. VI, No. 2 


Tas_e I. Comparison of Lake Erie and boiled University of Illinois water 


Lake Erie Water Boiled U. I. Well Water 

DE (COs) paket Beet ere 7.0 9.0 

GOr 5 Beka Sone ek bee ee 54 ppm. 250 ppm. 

SOG Say foe Rahn ae che eee pee 10 ppm. trace 

Oy MPA ao et att tat char hs fy Shite 5 ppm. I ppm. 

Cas Sher Sah een rs 27 ppm. 33 ppm. 

IMS aachote wicteca cee Ie ae tee ee 7 ppm. 27 ppm. 
Nasik oc) ie esi ce ce eee ee eee 5 ppm. 16 ppm. 
‘Lotal sods a, oo ttn, ve aoe eee 112 ppm. 248 ppm. 


TABLE II. Showing the development of whitefish eggs under different conditions 


Figures are days to stage indicated on left. Dates of spawning, receipt at Urbana, 
and beginning of experiment, respectively, follow each series number. Series II and 
III are stock 1; series VIII is stock 3. The entries in italics indicate that embryos died 
soon afterward. Oxygen all given in cc. per liter. 


A-I B-1 C-1 A-2 B-2 C-2 A-3 B-3 
Series II; 12/2; 
T2/39 12/6: 
DIderan Se emer meee 6.2-7.5| 5.8-8.9| 6.4-8.1| 6.4-8.0] 6.2-8.9] 7.9-8.5| 6.6-8.3| 6.8-9.2 
pHimea nine ges vee: 6.2 6.2 6.4 7.0 We We Fest » Oh ENG 
Oxygen range....... o—.9 0-1.04 | 0-2.08 | 0-.9 0-1.24 | .0I-3.7] 0-.9 O-1.7 
Oxygen average..... cD 2 6 is LZ a7. gi 3 
Cap small cells...... 4 4 4 4 4 4 4 4 
Embryo forming. ...| 5 5 5 5 5 5 5 5 
Post ring large...... 6 6 6 6 
Post ring small...... 7 7 7 ii 7 7 7 10 
Eye vesicle......... 
Pailtilat. fee see Io 10 9.5 IO 10 9 10 10.8 
ailestartin awe see 12 II rt 10 II 
Tail elongating...... 13 1g 13 II 
Tails, Doadye «:. eae 13 
Series III; 12/2; 
fofss 12/17, 
pHiiangers! ..cieere 5.9-6.9| 6.8-7.0] 6.8-9.0 cae 6.8- 7.0] 6.8-9.0 Be 6.8-7.0 
DHtMednt eee 6.4 8.5 6.4 8.5 6.4 
Oxygen range....... .4-2.4| .4-2.2] .2-1.7] 1.3-3.1| 1.9-3.7| I.7-3.2] 3.2-5.3] 3-3-4.9 
O: © ~eniaverage: ..... 1.64 .88 1.07 2.32 2.56 2.49 4.37 3.96 
i ‘igmented...... 15 15 15 15 15 15 15 15 
Fitrays!short. .2--.|19 19 7 IQ 17 17 19 17 
Fin rays 4 fin...... 21 21 21 
Tail near head...... 26-27. |20 26 26 26 
‘Dail totheadiver-es 27 27 27. 2 
Wail-toveyene erm 
Hatching<feeee es. 
Series. VIII; 12/7; 
D202) oy 
pH rangesaee ee eee 6.1-7.2|. 6.2-9.0| 9.0 6.1-7.2| 6.2-9.0] 9.0 6.1-7.2| 6.2-9.0 
DH meanest ce 6.4 eS 9.0 6.4 7.6 9.0 6.4 7.6 
Oxygen range....... OV siete Vanes 18—2°4/02,0—3°2|\ aa —oe5 2 0-30] asd oko eres 
Oxygen average..... 61 4 1.6 2.6 2.12 3.0 3.5 3.8 
Ready to hatch..... 76 76 76 76 76 76 76 76 
Hatching—tst...... 78, 7% |78, 13%|78, 18%|78, 18%|78, 16%|78, 15 %|78, 8% |78, 17% 


Max. hatching...... 79-82 |79-82 |79 81-83 |79-82 |79-81 {81-83 {81-83 


April, 1925 DEVELOPMENT OF THE WHITEFISH 108 


Effect of Conditions on the Different Stocks 


Table II shows the number of days required to reach the different stages 
in the various concentrations. The numbers and letters used below will be 
found in this table. 

Stock r. In series II, conditions varied materially in all dishes. With 
a pH around 6.2-6.4 we find that a set of eggs put into the dishes 4, B and 
C-1 on the seventh day varied in both amount of development and length of 
life roughly in proportion to the amount of oxygen supplied. This was 
I ce., .2 cc., .6 cc. per liter respectively. This amount was not enough to 
carry development beyond the elongation of the tail bud, and was lethal in 
every case. This same oxygen effect seemed to hold with higher pH, and 
was evidently below the threshold of whitefish development. 

In series III, oxygen content was raised so that dishes No. 1 had bagel 
I cc. per liter, dishes No. 2 about 2.5 cc., and dishes No. 3 about 4 cc. per 
liter. With this increase in oxygen concentration the more alkaline water 
showed the farthest development. The eggs lived as long in the acid and 
neutral waters of high and low oxygen but development was retarded. It 
is significant that those eggs in neutral waters were more retarded than those 
in either the acid or alkaline waters. Where neutrality was maintained as 
in this set a detrimental effect is noticed in development equal to that of 
higher hydrogen ion concentrations. The results suggest that pH of 7.8 is 
optimum and 7.0 too high a concentration of hydrogen ions. This lot of 
eggs, put in at the early pigmentation stage of the eyes lived nearly to hatch- 
ing. 

Stock 2. The experiments of series IV and V were started three days 
apart and run simultaneously so that conditions were identical except for the 
time of entering the experimental waters. The average oxygen for the 
different dishes was slightly higher than in series III. The eggs of series 
IV were put in when the fin buds were just starting. A distinct oxygen dif- 
ferential was established: the low oxygen eggs developed to the point where 
the tail reached the mendian line of the head; the medium oxygen eggs to the 
point where the tail reached past the head and around to the eye; while the 
high oxygen eggs developed just to hatching in the acid and neutral waters. 
The alkaline water did not seem enough lower in oxygen to account for the 
retardation which occurred, and this retardation was probably due to the 
hydrogen ion concentration. 

In series V more difference in development is apparent between the dif- 
ferent dishes. The greatest amount of retardation occurred in the low 
oxygen jars and in the high oxygen of the neutral series (pH fluctuating). 
Two eggs hatched in the medium oxygen of the neutral series, but all 
others died just before hatching time. These were the first individuals to 


109 ADA R. HALL Ecology, Vol. VI, No. 2 


hatch in the experimental dishes, though a few hatched in the stock (pH 7.8 
and oxygen 3.3 cc. per liter). 

Stock 3. Hatching occurred in the experiments run with both the third 
and fourth stocks. As these stocks were older, the time of exposure to the 
experimental waters was later, and a different reaction resulted. Series VI 
and VII were started two days apart but were run simultaneously. Hatch- 
ing occurred in several of the dishes and extended over several days, the 
beginning and duration of the process being recorded in Table II. The per 
cent hatching is given as the second member opposite ‘“‘ Hatching-Ist.” 

Series VIII was started just as the eggs of stock 3 were ready to hatch, 
to test the effect of different concentrations on this process and on the life of 
the larvae. Hatching occurred in all the dishes beginning on the same day. 
Maximum hatching was 2 days later in the acid “medium” oxygen and 
in the acid and neutral high oxygen, showing some retardation for these con- 
centrations. 

Stock 4. Series IX is the result of the exposure of eggs reared for the 
entire time of development at the hatchery in lake water. It shows the 
relative sensitivity of the hatching period and the newly emerged larvae. 
The first hatching occurred in the acid and neutral low oxygen an hour or 
two after putting the eggs in the water. A few also hatched in alkaline 
“low” and “ medium” oxygen later the same day. 


Differences in the Stocks at Hatching 


The normal time for the development of these eggs at the hatcheries was 
four to five months. Due to higher temperature my first stock began hatch- 
ing at thirty-two days. The newly hatched larvae from each of my four 
stocks were measured for body length, and for size and shape of yolk, and 
the time from hatching to death recorded. 

Only a few individuals were measured in stocks I and 2. In stock 1 the 
fry were 8 to 11 mm. in length with round yolk sacs 2 mm. in diameter. 
These lived only two days. In stock 2 the fry were 11 mm. long, also with 
round yolk sacs. These lived sixteen days with no food. Stock 3 averaged 
II.I mm. with similar yolks and lived at least ten days. Stock 4, raised in 
lake water, gave fry of a distinctly different type. They were 13.4 mm. to 
15 mm. long and were extremely active. The yolk sacs were oval but of the 
same volume as the earlier stocks. These lived for twenty-five days without 
food, and at the time of death measured 15 mm. This shows that eggs reared 
for a long period at low temperature attain a larger size and have more 
energy than they would have if forced at higher temperature (an argument 
against forcing conditions in hatchery work). 


April, 1925 DEVELOPMENT OF THE WHITEFISH 110 


Mortality 


Due to Fungus. One factor in mortality was fungus growth. This was 
in direct proportion to the amount of oxygen present, therefore was most 
detrimental in the high oxygen concentrations. The pH seemed to have no 
differential effect. The fungus grew very rapdily on dead eggs but also en- 
tangled live ones and killed them. Infected eggs left in the dishes would 
mat dozens of eggs together and kill them in a day or two. Since higher 
temperatures were more favorable to the fungus it grew more abundantly 
in my dishes than at the hatchery. 

Due to Experimental Conditions. The eggs of the different series (II to 
IX) were counted and the dead recorded from day to day. From this data 
the per cent of deaths was calculated for each dish of the series. The stages 
from early formation of the embryo and ring seemed very sensitive, as in all 
concentrations the deaths quickly amounted to 100 per cent. From the time 
of closure of the posterior ring and beginning of elongation of the tail and 
fin buds sensitivity decreased, as shown by the following figures: 


Series I]: Early gastrulation. At the end of three days the per cent which 
had died was as follows: A-1, 100 per cent; B-1 100 per cent; C-1, 66 
per cent; A=2) 100 per cent; B-=2, 75 per cent; C-2, 50 per cent; A-3, 
100 per cent; B-3, 95 per cent; C-3, 50 per cent. 

Series III: Eye pigmented, lens showing. At the end of nine days the per 
cent which had died was as follows: A-1, 20 per cent; B-1, 20 per cent; 
(=I, 10° per cent; A=2, 50: per cent; B-2/ 100 per, cent; C-2; 5° per 
cent; A-3, 10 per cent; B-3, 50 per cent; C-3, 5 per cent. 

Series IV: Tail 1/4 body length. At end of sixteen days the per cent which 
had died was as follows: A-1I, 91 per cent; B-1, 26 per cent; C-1, 49 
per cent; A-2, 64 per cent; B-2, 96 per cent; C-2, 98 per cent; A-3, 
70 per cent; B-3, 66 per cent; C-3, 98 per cent. 


The deaths due to fungus were in most cases small compared to total deaths. 
Total mortality is the index to the effect of varying conditions of oxygen and 
carbon dioxide, and shows the relative sensitivity of the different ages of 
eggs. 


Effect of Other Acids 


As sulphuric acid liberated the carbon dioxide in the above experiments, 
it was thought desirable to run a check with other acids. A series of finger 
bowls was prepared such that a pH of 6.3, 7.0, and 9.0 was obtained for each, 
sulphuric, hydrochloric, and acetic. Salts such as sodium chloride an- 
tagonize the acid which may be present in water (Loeb, ’12, Osterhout, 714), 
and therefore a duplicate series was made up in which one third of the volume 
of water was substituted by M/6 sodium chloride. The same amount of 


III ADA R. HALL Ecology, Vol. VI, No. 2 


mixing was given each solution as it was made up to insure a uniform oxygen 
content of 1.5 cc. per liter. The finger bowls were filled full and covered. 
Readings were taken without uncovering. Table III shows the time to death 
in the different concentrations. In the acetic and sulphuric acid (no salt) 
death occurred in pH 7.0 first, 9.0 second and 6.3 last, but for hydrochloric, 
death in 6.3 preceded 7.0. Neutrality was most quickly fatal, and alkalinity 
more toxic than acidity. The sulphuric plus salt was least toxic of all with a 
pH of 6.3, as these larvae were still alive at the end of 23 days. The differ- 


TABLE III. Resistance of whitefish larvae to acidity, neutrality, and alkalinity in 500 cc. 
fresh boiled water and M/6 NaCl water; acid, acetic 0.529N sulphuric 0.485N, 
hydrochloric 0.442N, COz gas added directly 


O2 in 


ee Chemical Used copnes pH Av. Time to Death Remarks 
4 | .8 cc. acetic + boiled H,O 1.5 6.3 47 hrs. 
41.8 CO ee ML OpNaGl ia eo Syke UE 
4 18cc. ‘* + boiled H2O * FAO 25e0- 
Ae AL SiCC eee NL OL Nal Lt 340 
4 | .6 cc. HCl + boiled HO es 6.3 20s 
ACTl..6) Coen t-te /6NaGl % ritstoy 
4 | .26cc. “ + boiled H.O 4 7.0 isp 
4 | .26cc. “* + M/6 NaCl % OTe es 
4 | .6 cc. HeSO,4 + boiled HO 2 6.3 Cee ik 
4 ees | PFE SE IM YAN ENG!| %E PAS) I alive 24 days 
4 |.16cc. ‘“ -+ boiled H,O is 7.0 ds 
4 6cc, ‘ + M/6 NaCl # cs 500 “ I alive 24 days 
4 | control boiled water z 9.0 ger 
4 le Oe Vi/GENaCl. beret See ie a: 87 ee 
24 | CO2 + boiled water af 6.3 2 hrs. 25 min. Stock 3 
45 “a ae ac Ai 6 zB 4 “ec “e 4 
15 | boiled water i G.0 bites ea See: Saas 
9 i‘ x Ai 9.0 Sees 
15 | COs + boiled water 2.8 6.4 5 ‘ (not dead) we gl 
18 ae os aé ae 2.8 6.4 12 “ec “ce “ec “ee 3 


ence in the reactions to the three acids was small. The variation in the 
hydrogen ions was probably influencing growth rather than the anions, sul- 
phate, acetate, and chloride. This is in keeping with the work of Loeb (’04, 
12) on the chemicals most influencing growth. He has studied the effect 
of salt solutions on growth and regeneration and finds that the cations or 
substituted hydrogen ions are most influential in changing growth, calcium, 
sodium, and potassium being necessary at some time during the life cycle for 
normal individuals to develop. 

The effect of direct addition of carbon dioxide to water of high and low 
oxygen was studied in relation to length of life in whitefish larvae. In low 
oxygen the larvae died more rapidly in acid water (6.3) than in alkaline 


April, 1925 DEVELOPMENT OF THE WHITEFISH 1I2 


(9.0). With high oxygen (3 cc. per liter) a pH of 6.3 did not kill, though 
the exposure was for the greater part of two days. This is in accordance 
with Well’s statement that large amounts of oxygen antagonize the detri- 
mental effects of high concentrations of carbon dioxide. Statements of the 
effect of the different combinations of oxygen and carbon dioxide in this 
paper seem contradictory. The experiments were all started with different 
stocks so the number of items that can be compared is unavoidably small. 
However when the time to “ fin ray short”’ (series III, table II) is plotted 
on cross section paper with oxygen and pH scales, and the points showing 
equal time connected in a manner suggested by Huntington’s (’19) plots of 
human death rate in relation to temperature and humidity, the results ap- 
pear orderly (Fig. 2). The time for “ tail flat” in series II shows a similar 


Oxygen in ce per liter 


Fic. 2. Showing equal time lines on an oxygen-hydrogen-ion chart. The broken 
line passes approximately through combinations of oxygen and hydrogen-ion concentra- 
tion in which the embryos developed a flat tail in ten days, beginning four days after 
spawning. The solid lines pass through combinations of oxygen and hydrogen-ion con- 
centration in which the embryos developed short fin rays in 17 and 19 days respectively. 
The general trend of these lines suggests that development may be expected to be most 
rapid at about 3.4 c.c. of oxygen and pH 7.6 to 7.7, where the large cross is placed. This 
cross is in the center of the ellipse. The center of the ellipse suggested by the flat tail 
curve would fall at a higher H-ion concentration. 


relation. Observations were not made often enough to bring out the neces- 
sary details in any but series III, as noted above; accordingly this interpreta- 
tion can be suggested only as a basis for further investigation. 


113 ADA R. HALL Ecology, Vol. VI, No. 2 


Under experimental conditions eggs develop into normal embryos with a 
pH of 7.0 and a much lower oxygen content than in the hatchery. Much 
greater variations were produced in per cent hatching and in length of time 
for development by varying the pH toward the acid or alkaline end of the 
scale than by changing the oxygen. This is in accord with the idea of 
Powers (’20). He concluded from his reconnaissance of the pH of Puget 
Sound waters under varying conditions of tides, weather, etc., that pH has 
more to do with compatibility of habitat than oxygen. 


Effect of Conditions on Reactions of Fry from Different Stocks 


As each of the stocks hatched, gradient experiments were run with the 
larvae to see if the environment during the early embryonic life had any ef- 
fect on the pH range which they would choose. A gradient tank suited to 
the size of the larvae was used. This tank was 20.5 cm. by 2 cm. by 1.5. cm. 
with screens 1.5 cm. from each end, and outlets at both sides of the middle. 
Water was introduced drop by drop behind the screens at the ends, and 
flowed out at the center. The end thirds were thus very nearly the same as 
the water introduced there, while the center third was a mixture of the two. 

' Stock 1 and 2 were reared from the early germinal cap stage and the 
elongation of the tail, respectively, in the vivarium waters. Both of these 
stocks then lived through the period of heart and blood formation in the ex- 
perimental waters. The results of the gradient experiments are suggestive. 
No larvae hatched from the experimental dishes of the first stock, but those 
hatching in the control (pH 7.8) stayed within the range of 7.6 to 8.2 when 
given a choice of 6.6 to 9.0. Fig. 3 A shows the reactions of larvae of stock 
2 to control water (7.8). When given a gradient of 6.4 to 8.2 a very definite 
choice of end is seen. Larvae reared in water of moderate hydroxyl ion 
concentration (pH 9.0, Fig. 2 B) choose the alkaline end of the tank, but 
contrary to expectation those from the high hydrogen ion experiments (pH 
6.4, C) also choose this end. None of the other larvae reared in any of the 
later stocks or experiments choose this low hydrogen ion concentration when 
there is water of a higher acidity available. 

In stock 4 the eggs were raised to hatching at Put-in-Bay and so began 
hatching a few hours after they were put into the experimental waters. 
These therefore passed the time of heart and blood formation in the lake 
water. Animals hatched on the 14th and 16th of March were kept in the 
experimental waters, and gradients run with them on the 17th and 22d. 
Nearly all of these showed the characteristic exploration of the whole tank 
for from three to five minutes then a preference for the acid end of the tank. 
The turning back occurred at a very definite pH and the avoidance was from 
either end, for often when an animal entered the unfavorable water it stayed 
there turning back from the changing point to again enter the unfavorable 


April, 1925 DEVELOPMENT OF THE WHITEFISH 114 


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Fic. 3. Reaction of larvae of the four stocks to a permanent gradient. A long, 
narrow tank was used with water of the desired pH entering at each end, making a 
permanent gradient for the entire period of the experiment. The fish swam freely in 
the tank for the time recorded in the marginal figures. A, control larva from stock 2. 
B, larva from stock 2 raised through the period of heart and blood formation in a pH 
of 8.6 to 9.0. C, larva from stock 2 raised through the period of heart and blood forma- 
tion in a pH of 6.4. D, larva of stock 4 raised in lake water, but hatched in pH of 6.4. 
E, larva of stock 4 hatched in a pH of 6.4 and given a choice of 8.0 io 9.0. 


115 ADA R. HALL Ecology, Vol. VI, No. 2 


end several times before it escaped to the more favorable water. We there- 
fore find the point of preference clearly marked. The fish hatched and 
kept in water of a pH of 6.3 show a preference for the acid end of a gradient 
(fig. 3 D). Those hatched and kept at 9.0 at first seemed to choose the 9.0 
end of the gradient but they gradually worked down into the acid end and 
stayed there. 

Wells (15) states that if a gradient is entirely confined to pH above 
neutrality, 8.0 to 9.0, that fish will choose the more alkaline end even though 
preferring an acid pH when given a choice of 6.4 to 9.0. I found that this 
was true of the whitefish larvae (fig. 3 EF). The larvae hatched in acid 
water (pH 6.4) chose more exclusively the part from 8.8 to 9.0 than those 
hatched in 9.0 water, although the latter turned at 8.4 and spent most of their 
time in the 9.0 end. Yet both would normally choose the acid end of a gradi- 
ent from 6.4 to 9.0. Wells believes that fish are avoiding pH 8.0, and indi- 
cates that if given a choice of pH 8.0 and 9.0 they will choose the latter. 
This seems to be a general reaction. Plankton studies of vertical distribu- 
tion show the smallest number of individuals at the thermocline (pH 8.0) 
with increasing numbers each side of it in either acid or alkaline waters. 

The whitefish larvae reared through the period of heart and blood forma- 
tion in experimental waters which were high in carbonate, and both quanti- 
tatively and qualitatively different in salt content generally from the lake 
waters from which the eggs were taken, behave in a gradient differently from 
fish reared in lake water but hatched in experimental waters. This difference 
lies in the direction of breaking up the preference, otherwise shown, for acid 
rather than alkaline waters, causing all larvae to prefer a pH of 7.2 to 8.2 
(regardless of the pH of the rearing waters). Gilbert (18) and Snyder 
((23) have shown that the salmon return to the tributary in which they were 
hatched. These experiments suggest that the chemical condition of the water 
during heart and blood formation modifies the reactions of fishes. The salt 
content and the hydrogen ion concentration of streams up which salmon run 
differ (Van Winkle ’14). These experiments suggest that the solution of 
these migration problems may be comparatively simple. 


Summary and Conclusions 


1. Hydrogen ion concentrations favorable for fertilization are too high 
for later development. Optimum hydrogen ion concentration is gradually 
lowered as the embryo becomes older. 

2. The most sensitive stages in whitefish development seem to be the 
first cleavages and early gastrulation. 

3. Fry hatching at one, two, and four months after spawning differ in 
size of body but not in size of yolk; those hatching at four months are 4 to 
6 mm. longer than those hatching earlier. The later fry also live longer than 
those hatching earlier, in spite of having the same amount of yolk available. 


April, 1925 DEVELOPMENT OF THE WHITEFISH 116 


4. Eggs raised through the period of heart and blood formation in water 
high in carbonates and differing from their normal environment show a dif- 
ferent type of gradient reaction to hydrogen ion concentrations. Such larvae 
raised in both acid (6.3) and alkaline water (9.0) choose the alkaline end of 
the gradient when 6.3 to 9.0 is offered. 

5. When exposed to water with a pH of 6.3 obtained by adding CO, di- 
rectly, the larvae died earlier in the acid water than in the alkaline with a low 
oxygen content. A high oxygen content antagonizes the CO, present, pro- 
longing the life of the larvae. 

6. In a gradient of 8.0 to 9.0 larvae of both acid and alkaline hatching 
environment choose the alkaline end (8.8 to 9.0). 

I wish to thank Dr. V. E. Shelford, at whose suggestion this work was 
undertaken, for his helpful advice and kindly criticism during the course of 
the investigation. 

The whitefish eggs were secured through the courtesy of the U. S. Bureau 
of Fisheries from the hatchery at Put-in-Bay. I wish to thank Mr. Downing 
and his associates for making my stay there both pleasant and profitable. 
The Bureau of Fisheries extended me the privileges at Put-in-Bay through 
Dr. H. B. Ward of the University of Illinois. 


LITERATURE CITED 


Clark, W. M. 1920. The determination of hydrogen ions. Baltimore. Williams and 
Wilkins. 

Clarke, F. W. 1920. The data of geochemistry. U. S. Geol. Survey Bull. 695. 

Cohn, E. J. 1918. Studies in the physiology of spermatazoa. Biol. Bull, 34: 167-218. 

Gilbert, C. H. 1918. Contributions to the life history of the sock-eye salmon (no. 5). 
Rep. Comm. of Fisheries of British Columbia, 1918. 

Hall, A. R. 1923. The effect of oxygen and carbon dioxide on the development of the 
toad (Bufo americanus LeConte). Ecology, 5: 290-310. 

Huntington, E. 1918. World power and evolution. Chapter V. New Haven. Yale 
University Press. 

Loeb, Jacques. 1904. On the influence of the reaction of the sea water on the regen- 
eration and growth of Tubularians. Univ. of Cal. Pub. Physiol., 1: 139-147. 

—. 1912. Mechanistic Conception of Life. Uniy. of Chicago Press. 

Osterhout, W. J. V. 1914. Antagonism between acids and salts. Jour. Biol. Chem., 
19: 517-520. 

Powers, E. B. 10920.° The variation of the condition of sea water, especially the hydro- 
gen ion concentration, and its relation to marine organisms. Pub. Pug. Sd. Biol. 
Sta., 2: 369-385. 

Shelford, V. E. 1918. Equipment for maintaining a flow of oxygen-free water and for 
controlling gas content. Bull. Ill. State Lab. Nat. Hist., 11: 573-575. 

Snyder, J. O. 1923. A second report on the return of salmon marked in 1915 in the 
Kalamath River. Calif. Fish and Game, 9: I-90. 

Wells, M. M. 10913. The resistance of fishes to different concentrations and combina- 
tions of oxygen and carbon dioxide. Biol. Bull., 25: 323-347. 

——. 1915. Reactions and resistance of fishes in their natural environment to acidity, 
alkalinity, and neutrality. Biol. Bull., 29: 221-237. 

Van Winkle, W. 1914. The quality of the surface waters of Washington. U. S. 
Geological Survey water supply paper No. 3309. 


VITA 


Ada Roberta Hall was born in Florida in 1890. She received her 
preparatory training in the Lincoln High School, Portland, Oregon, gradu- 
ating in 1908. She taught in country grade schools until February, 1911, 
when she entered the University of Oregon. During the period of her 
undergraduate work she taught the primary grades at Fern Ridge and 
Wendling, Oregon, for two years and a half, and was senior Playground 
Director in the City of Portland Parks for three summers. She was labora- 
tory assistant in Zoology at the University of Oregon during her senior year, 
and was a member of Scroll and Script, Senior Honorary Society. She 
received her A.B. degree with Highest Honors in Zoology in 1917. She 
continued at the University of Oregon for two years as graduate assistant 
in Zoology and received her A.M. degree iti 1919. The summer of 1918 was 
spent at the Puget Sound Biological Station, Friday Harbor, Washington. 
In the fall of 1919 she went to the University of Illinois as Fellow in Zo- 
ology. During the year 1920-21 she was elected to Sigma Xi and in the fall 
of 1920 to Iota Sigma Pi. The summer of 1920 was spent at the University 
of Michigan Biological Station at Douglas Lake. She completed her work 
for the degree of Doctor of Philosophy in June, 1921. During the period 
of graduate.study she published the following papers: 1. Some Experiments 
on the Resistance of Sea Urchin Eggs to Sulphurous Acid. Pub. Puget Sd. 
Biol. Sta. 2:113-119. 2. Regeneration in the Annelid Nerve Cord. Jour. 
Comp. Neur. vol. 33, No. 2:163-191. 


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